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Rapid, high efficiency isolation of pancreatic ß-cells.

ABSTRACT: The ability to isolate pure pancreatic ß-cells would greatly aid multiple areas of diabetes research. We developed a fluorescent exendin-4-like neopeptide conjugate for the rapid purification and isolation of functional mouse pancreatic ?-cells. By targeting the glucagon-like peptide-1 receptor with the fluorescent conjugate, ?-cells could be quickly isolated by flow cytometry and were >99% insulin positive. These studies were confirmed by immunostaining, microscopy and gene expression profiling on isolated cells. Gene expression profiling studies of cytofluorometrically sorted ?-cells from 4 and 12 week old NOD mice provided new insights into the genetic programs at play of different stages of type-1 diabetes development. The described isolation method should have broad applicability to the ?-cell field.

SUBMITTER: Clardy SM

PROVIDER: S-EPMC4557033 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Rapid, high efficiency isolation of pancreatic ß-cells.

Clardy Susan M SM Mohan James F JF Vinegoni Claudio C Keliher Edmund J EJ Iwamoto Yoshiko Y Benoist Christophe C Mathis Diane D Weissleder Ralph R

Scientific reports 20150902

The ability to isolate pure pancreatic ß-cells would greatly aid multiple areas of diabetes research. We developed a fluorescent exendin-4-like neopeptide conjugate for the rapid purification and isolation of functional mouse pancreatic β-cells. By targeting the glucagon-like peptide-1 receptor with the fluorescent conjugate, β-cells could be quickly isolated by flow cytometry and were >99% insulin positive. These studies were confirmed by immunostaining, microscopy and gene expression profiling ...[more]

PMID: 26330153

Similar Datasets

Rapid, high efficiency isolation of pancreatic ß-cells

Project description:The ability to isolate pure pancreatic ß-cells would greatly aid multiple areas of diabetes research. We developed an exendin-4-like neopeptide conjugate for the rapid purification and isolation of functional pancreatic ß-cells. By targeting the glucagon-like peptide-1 receptor, ß-cells were isolated within an hour and were >99% pure. These studies were confirmed by immunostaining, confocal microscopy and microarray analysis on isolated cells. Gene expression profiling studies of the cytofluorometrically sorted ß-cells provided new insights into the genetic programs at play of different ages and stages during type-1 diabetes development. The described isolation method should have broad applicability to the ß-cell field. Microarray profile of beta cells from isolated islets from 4 and 12 week old NOD mice. Cells were stained with 50 nM Ex4+ probe and sorted on FACS ARIA. DAPI and CD45+ cells were excluded.

2015-09-04 | E-GEOD-64508 | biostudies-arrayexpress

Rapid, high efficiency isolation of pancreatic ß-cells

2015-09-04 | GSE64508 | GEO

Rapid and High-Efficiency Laser-Alloying Formation of ZnMgO Nanocrystals.

Project description:Applications of ZnMgO nanocrystals (NCs), especially in photoelectric detectors, have significant limitations because of the unresolved phase separation in the synthesis process. Here, we propose a rapid and highly efficient ZnMgO NC alloying method based on pulsed laser ablation in liquid. The limit value of homogeneous magnesium (Mg) is pushed from 37% to 62%, and the optical band gap is increased to 3.7?eV with high doping efficiency (>100%). Further investigations on the lattice geometry of ZnMgO NCs indicate that all ZnMgO NCs are hexagonal wurtzite structures, and the (002) and (100) peaks shift to higher diffraction angles with the increase in Mg doping content. The calculated results of the lattice constants a and c slightly decrease based on Bragg's law and lattice geometry equations. Furthermore, the relationship between annealing temperature and the limit value of homogeneous Mg is examined, and the results reveal that the latter decreases with the former because of the phase separation of MgO. A probable mechanism of zinc magnesium alloy is introduced to expound on the details of the laser-alloying process.

| S-EPMC4914852 | biostudies-literature

Sequential Enzymatic Digestion of Different Cartilage Tissues: A Rapid and High-Efficiency Protocol for Chondrocyte Isolation, and Its Application in Cartilage Tissue Engineering.

Project description:ObjectiveThe classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering.DesignCartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity.ResultsOverall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol.ConclusionsThe sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.

| S-EPMC8804790 | biostudies-literature

Nuclear hormone retinoid X receptor (RXR) negatively regulates the glucose-stimulated insulin secretion of pancreatic ß-cells.

Project description:ObjectiveRetinoid X receptors (RXRs) are members of the nuclear hormone receptor superfamily and are thought to be key regulators in differentiation, cellular growth, and gene expression. Although several experiments using pancreatic ?-cell lines have shown that the ligands of nuclear hormone receptors modulate insulin secretion, it is not clear whether RXRs have any role in insulin secretion.Research design and methodsTo elucidate the function of RXRs in pancreatic ?-cells, we generated a double-transgenic mouse in which a dominant-negative form of RXR? was inducibly expressed in pancreatic ?-cells using the Tet-On system. We also established a pancreatic ?-cell line from an insulinoma caused by the ?-cell-specific expression of simian virus 40 T antigen in the above transgenic mouse.ResultsIn the transgenic mouse, expression of the dominant-negative RXR enhanced the insulin secretion with high glucose stimulation. In the pancreatic ?-cell line, the suppression of RXRs also enhanced glucose-stimulated insulin secretion at a high glucose concentration, while 9-cis-retinoic acid, an RXR agonist, repressed it. High-density oligonucleotide microarray analysis showed that expression of the dominant-negative RXR affected the expression levels of a number of genes, some of which have been implicated in the function and/or differentiation of ?-cells.ConclusionsThese results suggest that endogenous RXR negatively regulates the glucose-stimulated insulin secretion. Given these findings, we propose that the modulation of endogenous RXR in ?-cells may be a new therapeutic approach for improving impaired insulin secretion in type 2 diabetes.

| S-EPMC2963544 | biostudies-literature

Conditional gene targeting in mouse pancreatic ß-Cells: analysis of ectopic Cre transgene expression in the brain.

Project description:ObjectiveConditional gene targeting has been extensively used for in vivo analysis of gene function in ?-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate ?-cell- or pancreas-specific recombination, also drive Cre expression in the brain.Research design and methodsTransgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by ?-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1(tm1Cvw) lacZ knock-in mice. Cre expression in ?-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry.ResultsAll transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)(89.1Dam) mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)(1Lphi) mice were the only line that lacked Cre activity in the brain.ConclusionsCre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet ?-cells.

| S-EPMC2992770 | biostudies-literature

IL23 and TGF-ß diminish macrophage associated metastasis in pancreatic carcinoma.

Project description:The precise role of tumor associated macrophages remains unclear in pancreatic ductal adenocarcinoma (PDAC) while TGF-ß has an unclear role in metastases formation. In order to understand the role of IL23, an interleukin associated with macrophage polarization, we investigated IL23 in the context of TGF-ß expression in PDAC. We hypothesized that IL23 expression is associated with metastatic development and survival in PDAC. We investigated IL23 and TGF-ß protein expression on resected PDAC patient tumor sections who were divided into short-term (<12 months) survivors and long-term (>30 months) survivors. Panc-1 cells treated with IL23, TGF-ß, macrophages, or combinations thereof, were orthotopically implanted into NSG mice. Patients in the long-term survivor group had higher IL23 protein expression (P?=?0.01). IL23 expression was linearly correlated with TGF-ß expression in patients in the short-term survivor group (P?=?0.038). Macrophages induce a higher rate of PDAC metastasis in the mouse model (P?=?0.02), which is abrogated by IL23 and TGF-ß treatment (P?<?0.001). Macrophages serve a critical role in PDAC tumor growth and metastasis. TGF-ß contributes to a less tumorigenic TME through regulation of macrophages. Macrophages increases PDAC primary tumor growth and metastases formation while combined IL23 and TGF-ß pre-treatment diminishes these processes.

| S-EPMC5895618 | biostudies-literature

Rapid isolation of antigen-specific B-cells using droplet microfluidics.

Project description:Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusion inefficiency and display bias, respectively. Here we present a platform, featuring droplet microfluidics and a bead-based binding assay, to rapidly identify and verify antigen-binding antibody sequences from primary cells. We used a defined mixture of hybridoma cells to characterize the system, sorting droplets at up to 100 Hz and isolating desired hybridoma cells, comprising 0.1% of the input, with a false positive rate of less than 1%. We then applied the system to once-frozen primary B-cells to isolate rare cells secreting target-binding antibody. We performed RT-PCR on individual sorted cells to recover the correctly paired heavy- and light-chain antibody sequences, and we used rapid cell-free protein synthesis to generate single-chain variable fragment-format (scFv) antibodies from fourteen of the sorted cells. Twelve of these showed antigen-specific binding by ELISA. Our platform facilitates screening animal B-cell repertoires within days at low cost, increasing both rate and range of discovering antigen-specific antibodies from living organisms. Further, these techniques can be adapted to isolate cells based on virtually any secreted product.

| S-EPMC9055518 | biostudies-literature

High efficiency all-polymer tandem solar cells.

Project description:In this work, we have reported for the first time an efficient all-polymer tandem cell using identical sub-cells based on P2F-DO:N2200. A high power conversion efficiency (PCE) of 6.70% was achieved, which is among the highest efficiencies for all polymer solar cells and 43% larger than the PCE of single junction cell. The largely improved device performance can be mainly attributed to the enhanced absorption of tandem cell. Meanwhile, the carrier collection in device remains efficient by optimizing the recombination layer and sub-cell film thickness. Thus tandem structure can become an easy approach to effectively boost the performance of current all polymer solar cells.

| S-EPMC4881030 | biostudies-literature

Isolation and Characterization of High-Efficiency Rhizobia From Western Kenya Nodulating With Common Bean.

Project description:Common bean is one of the primary protein sources in third-world countries. They form nodules with nitrogen-fixing rhizobia, which have to be adapted to the local soils. Commercial rhizobial strains such as Rhizobium tropici CIAT899 are often used in agriculture. However, this strain failed to significantly increase the common bean yield in many places, including Kenya, due to the local soils' low pH. We isolated two indigenous rhizobial strains from the nodules of common bean from two fields in Western Kenya that have never been exposed to commercial inocula. We then determined their ability to fix nitrogen in common beans, solubilize phosphorus, and produce indole acetic acid. In greenhouse experiments, common bean plants inoculated with two isolates, B3 and S2 in sterile vermiculite, performed better than those inoculated with CIAT899 or plants grown with nitrogen fertilizer alone. In contrast to CIAT899, both isolates grew in the media with pH 4.8. Furthermore, isolate B3 had higher phosphate solubilization ability and produced more indole acetic acid than the other two rhizobia. Genome analyses revealed that B3 and S2 are different strains of Rhizobium phaseoli. We recommend fieldwork studies in Kenyan soils to test the efficacy of the two isolates in the natural environment in an effort to produce inoculants specific for these soils.

| S-EPMC8461304 | biostudies-literature

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